One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of the MAP kinases have been identified and each of these consists of a specific module of downstream kinases. One subgroup of the MAP kinases is the Jun N-terminal kinase/Stress activated protein kinase (JNK/SAPK) cascade. This pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation (Moriguchi et al., Adv. Pharmacol., 1996, 36, 121-137; Su and Karin, Curr. Opin. Immunol., 1996, 8, 402-411).
Jun N-terminal Kinase Kinase-1 (also known as JNKK1, PRKMK4, MEK4, MKK4, SAPK/Erk kinase 1 and SEK1) is a dual-specificity MAP kinase kinase that acts to mediate signaling pathways initiated by cellular stressors (Derijard et al., Science, 1995, 267, 682-685). Jun N-terminal Kinase Kinase-1 has been shown to be regulated and activated to varying degrees by ultraviolet exposure (Butterfield et al., Biochem. J., 1999, 338, 681-686), the .beta..gamma. subunit of G-proteins (Yamauchi et al., J. Biol. Chem., 1999, 274, 1957-1965) and mixed lineage kinase-2 (Cuenda and Dorow, Biochem. J., 1998, 333, 11-15; Hirai et al., J. Biol. Chem., 1998, 273, 7406-7412) suggesting that only certain stresses affect Jun N-terminal Kinase Kinase-1-mediated signaling pathways. Further investigations into the role of Jun N-terminal Kinase Kinase-1 in MAP kinase signaling cascades demonstrated that the interaction of Jun N-terminal Kinase Kinase-1 with both upstream and downstream components in the JNK signaling pathway is mediated through the amino terminus of the protein (Xia et al., Genes Dev., 1998, 12, 3369-3381).
Three isoforms of Jun N-terminal Kinase Kinase-1 have been isolated and these are disclosed in U.S. Pat. Nos. 5,736,381 and 5,804,427. Also disclosed are polynucleotides that encode three isoforms of Jun N-terminal Kinase Kinase-1, expression vectors comprising the polynucleotide sequence and host cells transformed with the recombinant expression vector as well as the Jun N-terminal Kinase Kinase-1 polypeptide encoded by the polynucleotide (Davis et al., 1998; Davis et al., 1998).
Recently it was reported that Jun N-terminal Kinase Kinase-1 may act as a tumor suppressor gene, and sequence variants resulting from mutations and mismatches have been found in certain types of cancers including pancreatic, lung, breast and colon cancer cells (Su et al., Cancer Res., 1998, 58, 2339-2342; Teng et al., Cancer Res., 1997, 57, 4177-4182).
Disruption of the Jun N-terminal Kinase Kinase-1 gene in mice by homologous recombination resulted in embryos that displayed severe anemia and died by day 10.5. It was noted that, although hematopoiesis was normal, hepatogenesis and liver formation were severely impaired, with liver cells undergoing massive apoptosis and defects in AP-1 transcriptional activity (Ganiatsas et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 6881-6886; Nishina et al., Development, 1999, 126, 505-516; Yang et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 3004-3009). Characterization of chimeric mice lacking Jun N-terminal Kinase Kinase-1 showed that mutation of Jun N-terminal Kinase Kinase-1 did not alter the induction of apoptosis in response to metabolic poisons but that T-cells failed to induce the expression of the death suppressor, Bcl-XL, suggesting that Jun N-terminal Kinase Kinase-1 is required to transduce cell-survival signals (Nishina et al., J. Immunol., 1998, 161, 3416-3420).
To date, strategies aimed at inhibiting Jun N-terminal Kinase Kinase-1 function have involved the use of antibodies, dominant-negative forms of Jun N-terminal Kinase Kinase-1, gene knockouts in mice and SAPK pathway inhibitors. Disclosed in the PCT application WO 98/54203 are inhibitors of the SAPK pathway comprising antisense oligonucleotides to an upstream kinase, MEKK1, as well as antisense oligonucleotides targeting the kinase isoforms that lie downstream of Jun N-terminal Kinase Kinase-1, SAPK1-3. Also disclosed are methods to inhibit the SAPK pathway using ribozymes, small molecule inhibitors and dominant-negative mutants (Mercola, 1998).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Jun N-terminal Kinase Kinase-1 and consequently, there remains a long felt need for additional agents capable of specifically inhibiting Jun N-terminal Kinase Kinase-1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Jun N-terminal Kinase Kinase-1 expression.